Experiments were initiated to characterize cell-mediated immune responses to human parainfluenza virus type 3 (PIV3). BALB/c mice were infected intranasally with RSV and splenocytes were isolated 3 to 6 weeks later. Upon stimulation in vitro with PIV3, cytolytic activity was demonstrated which was PIV3-specific, MHC class I-restricted and CD8 positive. The antigen specificities of these cytotoxic T cells (CTL) were investigated using recombinant vaccinia viruses which express the PIV3 hemagglutinin-neuraminidase (HN) glycoprotein, fusion (F) glycoprotein or major nucleocapsid (NP) protein (vac-HN, vac-F, or vac-NP). In studies to date, vac-HN and vac-F were tested for the ability to induce CTL upon intraperitoneal immunization, or for the ability to prepare targets in vitro for lysis by CTL from PIV3-infected mice. Neither vac-HN nor vac-F were effective in inducing PIV3-specific CTL, and neither recombinant was effective in preparing targets in vitro. Vac-NP remains to be tested. These results suggest that the HN and F glycoproteins, which are the major neutralization and protective antigens, are not significant CTL antigens in the BALB/c mouse. Presumably, one or more other viral proteins are responsible for inducing PIV3-specific CTLs, as will be elucidated in further studies using additional PIV3-vaccinia virus recombinants. This will form the basis for further studies to evaluate the relative abilities of CTL versus neutralizing antibodies to restrict PIV3 infection in the mouse model.